A “CRISPR” non-viral manufacturing approach for CAR T cell therapies
Authors: Matthew J. Cortese, Craig Sauter
Journal: Molecular Therapy (2022)

Chimeric antigen receptor (CAR) T cell therapies have developed over the past decade to become an increasingly important treatment for cancers, especially relapsed/refractory lymphoid malignancies. While promising, currently approved CAR T cell therapy products have limitations, including cost, consistency, and speed of manufacturing, toxicities including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and prolonged cytopenias. Moreover, in the common indication for relapsed and refractory large B cell lymphoma (LBCL), the majority of patients experience progression of disease (POD) following CAR T cell therapy. Those working to develop next-generation CAR T cell products are seeking to address these deficiencies, with a notable such effort by Zhang et al. recently published in Nature. 1 Zhang J. Hu Y. Yang J. Li W. Zhang M. Wang Q. Zhang L. Wei G. Tian Y. Zhao K. et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature. 2022; 609: 369-374 Crossref PubMed Scopus (44) Google Scholar Zhang et al. manufactured CAR T cells using a new non-viral, gene-specific integration approach using CRISPR-Cas9 introduced via electroporation. They subsequently knocked out several genes of interest by inserting a 4-1BB and CD3ζ CAR into the target locus, most notably PD1. These PD1-19bbz cells outperformed a CAR T cell product similar to current FDA-approved therapies that utilize lentiviral CAR transduction in an in vitro assay and were then tested in a small (8 patient) phase I clinical trial, demonstrating promising efficacy. These promising data were made more impressive by the fact that relatively small cell doses were used, specifically 560,000 cells/kg, which is compatible with low doses used in FDA-approved products (shown in Table 1). This is likely in part due to the high percentage of memory CAR T cells generated. This work is potentially transformational by offering a new CRISPR-Cas9-based CAR T cell manufacturing process that can edit out an undesirable target gene while enabling greater T cell persistence and efficacy. The phase I clinical trial only included 8 patients, and longer-term follow up will be needed to fully validate the findings. Table 1Current FDA-approved chimeric antigen receptor (CAR) T cell products + PD1-19bbz CAR T product name (trade name) Approved indications Registration trials (year, indication) and key efficacy outcomes Registration trial: Toxicity outcomes CAR T properties Axicabtagene ciloleucel (YESCARTA) R/R PMBCL; R/R HGBL; R/R DLBCL (+transformed); R/R FL (3L+) ZUMA-1 (2017, R/R PMBCL, HGBL, and DLBCL, FL grade 3B): 108 treated patients; ORR 83%, CRR 58%, mDOR 11.1 months, mPFS 5.9 months, mOS NR at 2 year follow-up; similar real-world dataZUMA-5 (2021, R/R FL, MZL): 104 treated patients eligible for analysis (84 FL, 20 MZL); ORR 92%, CRR 74%, mDOR NR (11.1 month MZL), mPFS NR (12 month MZL), mOS NR with 17.5 month median follow-upZUMA-7 (2022, R/R DLBCL in 2nd line compared with autologous BMT): 170 patients treated with Axi-cel ORR 83%, CRR 65%. mPFS 14.7 mos, mOS NR, mEFS 8.3 mos with 24.9 mos median follow-up ZUMA-1: all grade ≥3 AEs 48%; 18% cytopenias >3 months, CRS 11%, NEs 32%; TRM ∼2%.ZUMA-5: all grade ≥3 AEs 50%; cytopenias 70%, infection 18%, CRS 7%, NEs 19%; TRM 3% - CD19 targeted- lentiviral transduction- CD28 costim- 17 day median manufacturing time- cell dose: 2–200 million cells/kg Brexucabtagene autoleucel (TECARTUS) R/R MCL; R/R B-ALL (adults) ZUMA-2 (2020, MCL): 68 treated patients; ORR 91%, CRR 68%, mDOR 28.2 months, mPFS 25.8 months, mOS 46.6 months with 35.6 month median follow-upZUMA-3 (2021, B-ALL): 45 treated patients; ORR 75%, CRR 69%, mDOR 14.5 months, mPFS 7.3 months, mOS 12.1 months with 22.1 month median follow-up ZUMA-2: grade ≥3 cytopenias 94%; infection 32%, CRS 15%, NEs 31%; TRM ∼3%ZUMA-3: grade ≥3 CRS 31%, NEs 38%; TRM ∼4% - CD19 targeted- lentiviral transduction- CD28 costim- 15–16 day median manufacturing time- cell dose: 2–200 million cells/kg Lisocabtagene maraleucel (BREYANZI) R/R DLBCL (+transformed); R/R HGBL; R/R PBMCL; R/R FL grade 3B TRANSCEND (2021, R/R DLBCL, HGBL, PBMCL, FL grade 3B): 269 treated patients, ORR 73%, CRR 53%, mOS 18.8 monthsTRANSFORM: 92 treated patients, mEFS 10.1 months at 6.2 month median follow-up TRANSCEND: grade ≥3 neutropenia 60%, anemia 37%, thrombocytopenia 27%, CRS 2%, NEs 10%; TRM <1%TRANSFORM: similar to TRANSCEND - CD19 targeted- lentiviral transduction- 4-1BB costim- 26 day median manufacturing time- cell dose: 50–110 million cells Tisagenlecleucel (KYMRIAH) R/R DLBCL; R/R FL (3L+); R/R B-ALL age ≤25 years JULIET (2018, R/R DLBCL): 93 treated patients; ORR 52%, 40% CRR, 12 months PFS 65% (79% with prior CR)ELARA (2022, R/R FL): 97 treated patients; ORR 86.2%, CRR 69.1%, median follow-up of 16.59 monthsCCTL019B2202 (2017, R/R ALL): CRR 63%, median follow-up of 4.8 months JULIET: grade ≥3 CRS 22%, NEs 12%, cytopenias >28 days 32%, infections 20%, febrile neutropenia 14%; 0% TRM but 3 deaths from PD within 30 days.ELARA: 48.5% CRS (0% grade ≥3), NEs 37.1% (3% grade ≥3); no TRMCCTL019B2202: CRS 79%, NEs 65% - CD19 targeted- lentiviral transduction- 4-1BB costim- 54 day median manufacturing time- cell dose: 600,000 to 600 million cells Idecabtagene vicleucel (ABECMA) R/R MM (5L + after prior immunomodulatory agent, proteasome inhibitor, and an anti-CD38 mAB) KarMMa (2021, R/R MM): 128 treated patients; ORR 73%, CRR 33%, MRD-negative rate 26%, mDOR 10.7 months, mPFS 8.8 months, mOS 19.4 months with median follow-up of 13.3 months KarMMa: grade ≥3 neutropenia 89%, anemia 60%, thrombocytopenia 52%, infection 22%, CRS 4% (84% overall), 3% NEs (18%); ∼3% TRM - BCMA targeted- lentiviral transduction- 4-1BB costim- 15 day median manufacturing time- cell dose: 300–460 million cells Ciltacabtagene autoleucel (CARVYKTI) R/R MM (5L + after prior immunomodulatory agent, proteasome inhibitor, and an anti-CD38 mAB) CARTITUDE-1 (2022, R/R MM): 97 treated patients; ORR 97%, CRR 67%, mDOR NR, mPFS N with 12 months PFS 77% and OS 89% with median follow-up of 12.4 months CARTITUDE-1: grade ≥3 neutropenia 95%, anemia 68%, thrombocytopenia 60%, CRS 4% (95% overall), NEs 9% (21% overall); ∼6% TRM - BCMA targeted- lentiviral transduction- 4-1BB costim- 29 day median manufacturing time- cell dose: 500,000 to 1 million cells/kg PD1-19bbz (Zhang et al. 1 Zhang J. Hu Y. Yang J. Li W. Zhang M. Wang Q. Zhang L. Wei G. Tian Y. Zhao K. et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature. 2022; 609: 369-374 Crossref PubMed Scopus (44) Google Scholar ) N/A “Non-viral, specifically targeted CAR T cells achieve high safety and efficacy in B-NHL” (2022, aggressive B-NHL); 8 patients treated, ORR 100%, CRR 87.5% (62.5% durable) with median follow-up of 12 months Grade <3 CRS in 50% (none grade ≥3), no NEs, transient/low-grade cytopenias with chemotherapy - CD19 + PD1 knockout targeted- CRISPR-Cas9 electroporation (non-viral)- cell dose: 560,000 to 2.35 million/kg R/R, relapsed/refractory; diffuse large B cell lymphoma, DLBCL; B-ALL, B cell acute lymphoblastic leukemia; PMBCL, primary mediastinal large B cell lymphoma; HGBL, high-grade B cell lymphomas; FL, follicular lymphoma; #L+, indicated line of therapy or later; MCL, mantle cell lymphoma; MM, multiple myeloma; MZL, marginal zone lymphoma; ORR, overall response rate; CRR, complete response rate; mDOR, median duration of remission; mPFS, median progression-free survival; mOS, median overall survival; AE, adverse event; CRS, cytokine release syndrome; NEs, neurologic events; TRM, treatment-related mortality; MRD, minimal residual disease. Open table in a new tab R/R, relapsed/refractory; diffuse large B cell lymphoma, DLBCL; B-ALL, B cell acute lymphoblastic leukemia; PMBCL, primary mediastinal large B cell lymphoma; HGBL, high-grade B cell lymphomas; FL, follicular lymphoma; #L+, indicated line of therapy or later; MCL, mantle cell lymphoma; MM, multiple myeloma; MZL, marginal zone lymphoma; ORR, overall response rate; CRR, complete response rate; mDOR, median duration of remission; mPFS, median progression-free survival; mOS, median overall survival; AE, adverse event; CRS, cytokine release syndrome; NEs, neurologic events; TRM, treatment-related mortality; MRD, minimal residual disease.